Abstract
A conventional microscope produces a sharp image from just a single object-plane. This is often a limitation, notably in cell biology. We present a microscope attachment which records sharp images from several object-planes simultaneously. The key concept is to introduce a distorted diffraction grating into the optical system, establishing an order-dependent focussing power in order to generate several images, each arising from a different object-plane. We exploit this multiplane imaging not just for bio-imaging but also for nano-particle tracking, achieving approximately 10 nm z position resolution by parameterising the images with an image sharpness metric.
Highlights
High resolution optical microscopy is a vital analytical tool in cell biology as it offers a noninvasive and non-destructive diagnostic technique [1,2]
A conventional microscope produces a sharp image from just a single object-plane
The two main 3D imaging techniques are scanning confocal microscopy and wide-field imaging [1]. 3D imaging in a confocal system is achieved by scanning the laser spot relative to the sample
Summary
High resolution optical microscopy is a vital analytical tool in cell biology as it offers a noninvasive and non-destructive diagnostic technique [1,2]. 3D imaging in a confocal system is achieved by scanning the laser spot relative to the sample. The two main 3D imaging techniques are scanning confocal microscopy and wide-field imaging [1]. It gives excellent sectioning properties by rejecting out-of-focus light but scanning limits the imaging speed. High resolution in all three directions is achieved only in a small region around the focal plane. These techniques do not yield a 3D image of the host cell which may be changing continuously
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